Leucine-rich Repeat Kinase 2 Kinase Inhibitors Research Articles

Macrophage LRRK2 hyperactivity impairs autophagy and induces Paneth cell dysfunction.

LRRK2 polymorphisms (G2019S/N2081D) that increase susceptibility to Parkinson's disease and Crohn's disease (CD) lead to LRRK2 kinase hyperactivity and suppress autophagy. This connection suggests that LRRK2 kinase inhibition, a therapeutic strategy being explored for Parkinson's disease, may also benefit patients with CD. Paneth cell homeostasis is tightly regulated by autophagy, and their dysfunction is a precursor to gut inflammation in CD. Here, we found that patients with CD and mice carrying hyperactive LRRK2 polymorphisms developed Paneth cell dysfunction. We also found that LRRK2 kinase can be activated in the context of interactions between genes (genetic autophagy deficiency) and the environment (cigarette smoking). Unexpectedly, lamina propria immune cells were the main intestinal cell types that express LRRK2, instead of Paneth cells as previously suggested. We showed that LRRK2-mediated pro-inflammatory cytokine release from phagocytes impaired Paneth cell function, which was rescued by LRRK2 kinase inhibition through activation of autophagy. Together, these data suggest that LRRK2 kinase inhibitors maintain Paneth cell homeostasis by restoring autophagy and may represent a therapeutic strategy for CD.

LRRK2 Inhibitors as Promising Treatment for Parkinson's Disease.

Parkinson's disease (PD) is one of the most prevalent neurodegenerative disorders, with current treatments offering only temporary symptomatic relief. There is an urgent need for the development of novel therapeutic approaches. Abnormal increases in LRRK2 kinase activity have been identified in both sporadic and familial PD patients, suggesting that inhibiting LRRK2 kinase activity presents a promising avenue for the pursuit of effective PD treatment strategies. In this Viewpoint, we discuss the exciting new insights regarding the development of LRRK2 kinase inhibitors as a treatment for Parkinson's disease.

LRRK2 regulates production of reactive oxygen species in cell and animal models of Parkinson's disease.

Oxidative stress has long been implicated in Parkinson's disease (PD) pathogenesis, although the sources and regulation of reactive oxygen species (ROS) production are poorly defined. Pathogenic mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are associated with increased kinase activity and a greater risk of PD. The substrates and downstream consequences of elevated LRRK2 kinase activity are still being elucidated, but overexpression of mutant LRRK2 has been associated with oxidative stress, and antioxidants reportedly mitigate LRRK2 toxicity. Here, using CRISPR-Cas9 gene-edited HEK293 cells, RAW264.7 macrophages, rat primary ventral midbrain cultures, and PD patient-derived lymphoblastoid cells, we found that elevated LRRK2 kinase activity was associated with increased ROS production and lipid peroxidation and that this was blocked by inhibitors of either LRRK2 kinase or NADPH oxidase 2 (NOX2). Oxidative stress induced by the pesticide rotenone was ameliorated by LRRK2 kinase inhibition and was absent in cells devoid of LRRK2. In a rat model of PD induced by rotenone, a LRRK2 kinase inhibitor prevented the lipid peroxidation and NOX2 activation normally seen in nigral dopaminergic neurons in this model. Mechanistically, LRRK2 kinase activity was shown to regulate phosphorylation of serine-345 in the p47phox subunit of NOX2. This, in turn, led to translocation of p47phox from the cytosol to the membrane-associated gp91phox (NOX2) subunit, activation of the NOX2 enzyme complex, and production of ROS. Thus, LRRK2 kinase activity may drive cellular ROS production in PD through the regulation of NOX2 activity.

New Pyrrolopyrimidines as LRRK2 Inhibitors for Treating Parkinson's Disease.

This patent describes the novel pyrroloppyrimidine compounds as LRRK2 kinase inhibitors. The patent includes the synthesis of compounds, compositions containing them and their use in the treatment of or prevention of diseases associated with LRRK2 kinase activity, such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS).

Targeting Specific Kinase Substrates Rescues Increased Colitis Severity Induced by the Crohn's Disease-Linked LRRK2-N2081D Variant.

LRRK2 contains a kinase domain where both the N2081D Crohn's disease (CD) risk and the G2019S Parkinson's disease (PD)-pathogenic variants are located. The mechanisms by which the N2081D variant increase CD risk, and how these adjacent mutations result in distinct diseases, remain unclear. To investigate the pathophysiology of the CD-linked LRRK2 N2081D variant, we generated a knock-in (KI) mouse model and compared its effects to those of the LRRK2-G2019S mutation. We find that Lrrk2 N 2081 D KI mice demonstrate heightened sensitivity to induced colitis, resulting in more severe inflammation and intestinal damage than Lrrk2 G2019S KI and wild-type mice. Analysis of Colon tissue revealed distinct mutation-dependent LRRK2 RAB substrate phosphorylation, with significantly elevated phosphorylated RAB10 levels in Lrrk2 N2081D mice. In cells, we demonstrate that the N2081D mutation activates LRRK2 through a mechanism distinct from that of LRRK2-G2019S. We further find that proinflammatory stimulation enhances LRRK2 kinase activity, leading to mutation-dependent differences in RAB phosphorylation and inflammatory responses in dendritic cells. Finally, we show that genetic knockout of Rab12 , but not pharmacological LRRK2 kinase inhibition, significantly reduced colitis severity in Lrrk2 N2081D mice. Our study characterizes the pathogenic mechanisms of LRRK2-linked CD, highlights important structural and functional differences between disease-associated LRRK2 variants, and suggests RAB proteins as promising therapeutic targets for modulating LRRK2 activity in CD treatment.

A designed ankyrin-repeat protein that targets Parkinson’s disease-associated LRRK2

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson’s disease (PD) has led to intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

LRRK2 Kinase Inhibitor PF-06447475 Protects Drosophila melanogaster against Paraquat-Induced Locomotor Impairment, Life Span Reduction, and Oxidative Stress

Parkinson’s disease (PD) is a complex multifactorial progressive neurodegenerative disease characterized by locomotor alteration due to the specific deterioration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc). Mounting evidence shows that human LRRK2 (hLRRK2) kinase activity is involved in oxidative stress (OS)-induced neurodegeneration, suggesting LRRK2 inhibition as a potential therapeutic target. We report that the hLRRK2 inhibitor PF-06447475 (PF-475) prolonged lifespan, increased locomotor activity, maintained DAergic neuronal integrity, and reduced lipid peroxidation (LPO) in female Drosophila melanogaster flies chronically exposed to paraquat (PQ), a redox cycling compound, compared to flies treated with vehicle only. Since LRRK2 is an evolutionary conserved kinase, the present findings reinforce the idea that either reduction or inhibition of the LRRK2 kinase might decrease OS and locomotor alterations associated with PD. Our observations highlight the importance of uncovering the function of the hLRRK2 orthologue dLrrk2 in D. melanogaster as an excellent model for pharmacological screenings.

LRRK2 kinase inhibition protects against Parkinson's disease-associated environmental toxicants

Idiopathic Parkinson's disease (PD) is epidemiologically linked with exposure to toxicants such as pesticides and solvents, which comprise a wide array of chemicals that pollute our environment. While most are structurally distinct, a common cellular target for their toxicity is mitochondrial dysfunction, a key pathological trigger involved in the selective vulnerability of dopaminergic neurons. We and others have shown that environmental mitochondrial toxicants such as the pesticides rotenone and paraquat, and the organic solvent trichloroethylene (TCE) appear to be influenced by the protein LRRK2, a genetic risk factor for PD. As LRRK2 mediates vesicular trafficking and influences endolysosomal function, we postulated that LRRK2 kinase activity may inhibit the autophagic removal of toxicant damaged mitochondria, resulting in elevated oxidative stress. Conversely, we suspected that inhibition of LRRK2, which has been shown to be protective against dopaminergic neurodegeneration caused by mitochondrial toxicants, would reduce the intracellular production of reactive oxygen species (ROS) and prevent mitochondrial toxicity from inducing cell death. To do this, we tested in vitro if genetic or pharmacologic inhibition of LRRK2 (MLi2) protected against ROS caused by four toxicants associated with PD risk – rotenone, paraquat, TCE, and tetrachloroethylene (PERC). In parallel, we assessed if LRRK2 inhibition with MLi2 could protect against TCE-induced toxicity in vivo, in a follow up study from our observation that TCE elevated LRRK2 kinase activity in the nigrostriatal tract of rats prior to dopaminergic neurodegeneration. We found that LRRK2 inhibition blocked toxicant-induced ROS and promoted mitophagy in vitro, and protected against dopaminergic neurodegeneration, neuroinflammation, and mitochondrial damage caused by TCE in vivo. We also found that cells with the LRRK2 G2019S mutation displayed exacerbated levels of toxicant induced ROS, but this was ameliorated by LRRK2 inhibition with MLi2. Collectively, these data support a role for LRRK2 in toxicant-induced mitochondrial dysfunction linked to PD risk through oxidative stress and the autophagic removal of damaged mitochondria.

Suppression of Glioblastoma Stem Cell Potency and Tumor Growth via LRRK2 Inhibition.

Leucine-rich repeat kinase 2 (LRRK2), a large GTP-regulated serine/threonine kinase, is well-known for its mutations causing late-onset Parkinson's disease. However, the role of LRRK2 in glioblastoma (GBM) carcinogenesis has not yet been fully elucidated. Here, we discovered that LRRK2 was overexpressed in 40% of GBM patients, according to tissue microarray analysis, and high LRRK2 expression correlated with poor prognosis in GBM patients. LRRK2 and stemness factors were highly expressed in various patient-derived GBM stem cells, which are responsible for GBM initiation. Canonical serum-induced differentiation decreased the expression of both LRRK2 and stemness factors. Given that LRRK2 is a key regulator of glioma stem cell (GSC) stemness, we developed DNK72, a novel LRRK2 kinase inhibitor that penetrates the blood-brain barrier. DNK72 binds to the phosphorylation sites of active LRRK2 and dramatically reduced cell proliferation and stemness factors expression in in vitro studies. Orthotopic patient-derived xenograft mouse models demonstrated that LRRK2 inhibition with DNK72 effectively reduced tumor growth and increased survival time. We propose that LRRK2 plays a significant role in regulating the stemness of GSCs and that suppression of LRRK2 kinase activity leads to reduced GBM malignancy and proliferation. In the near future, targeting LRRK2 in patients with high LRRK2-expressing GBM could offer a superior therapeutic strategy and potentially replace current clinical treatment methods.

Opposing actions of JIP4 and RILPL1 provide antagonistic motor force to dynamically regulate membrane reformation during lysosomal tubulation/sorting driven by LRRK2.

Lysosomes are dynamic cellular structures that adaptively remodel their membrane in response to stimuli, including membrane damage. We previously uncovered a process we term LYTL (LYsosomal Tubulation/sorting driven by Leucine-Rich Repeat Kinase 2 [LRRK2]), wherein damaged lysosomes generate tubules sorted into mobile vesicles. LYTL is orchestrated by the Parkinson's disease-associated kinase LRRK2 that recruits the motor adaptor protein and RHD family member JIP4 to lysosomes via phosphorylated RAB proteins. To identify new players involved in LYTL, we performed unbiased proteomics on isolated lysosomes after LRRK2 kinase inhibition. Our results demonstrate that there is recruitment of RILPL1 to ruptured lysosomes via LRRK2 activity to promote phosphorylation of RAB proteins at the lysosomal surface. RILPL1, which is also a member of the RHD family, enhances the clustering of LRRK2-positive lysosomes in the perinuclear area and causes retraction of LYTL tubules, in contrast to JIP4 which promotes LYTL tubule extension. Mechanistically, RILPL1 binds to p150Glued, a dynactin subunit, facilitating the transport of lysosomes and tubules to the minus end of microtubules. Further characterization of the tubulation process revealed that LYTL tubules move along tyrosinated microtubules, with tubulin tyrosination proving essential for tubule elongation. In summary, our findings emphasize the dynamic regulation of LYTL tubules by two distinct RHD proteins and pRAB effectors, serving as opposing motor adaptor proteins: JIP4, promoting tubulation via kinesin, and RILPL1, facilitating tubule retraction through dynein/dynactin. We infer that the two opposing processes generate a metastable lysosomal membrane deformation that facilitates dynamic tubulation events.

LRRK2 kinase inhibition reverses G2019S mutation-dependent effects on tau pathology progression

BackgroundMutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease (PD). These mutations elevate the LRRK2 kinase activity, making LRRK2 kinase inhibitors an attractive therapeutic. LRRK2 kinase activity has been consistently linked to specific cell signaling pathways, mostly related to organelle trafficking and homeostasis, but its relationship to PD pathogenesis has been more difficult to define. LRRK2-PD patients consistently present with loss of dopaminergic neurons in the substantia nigra but show variable development of Lewy body or tau tangle pathology. Animal models carrying LRRK2 mutations do not develop robust PD-related phenotypes spontaneously, hampering the assessment of the efficacy of LRRK2 inhibitors against disease processes. We hypothesized that mutations in LRRK2 may not be directly related to a single disease pathway, but instead may elevate the susceptibility to multiple disease processes, depending on the disease trigger. To test this hypothesis, we have previously evaluated progression of α-synuclein and tau pathologies following injection of proteopathic seeds. We demonstrated that transgenic mice overexpressing mutant LRRK2 show alterations in the brain-wide progression of pathology, especially at older ages.MethodsHere, we assess tau pathology progression in relation to long-term LRRK2 kinase inhibition. Wild-type or LRRK2G2019S knock-in mice were injected with tau fibrils and treated with control diet or diet containing LRRK2 kinase inhibitor MLi-2 targeting the IC50 or IC90 of LRRK2 for 3–6 months. Mice were evaluated for tau pathology by brain-wide quantitative pathology in 844 brain regions and subsequent linear diffusion modeling of progression.ResultsConsistent with our previous work, we found systemic alterations in the progression of tau pathology in LRRK2G2019S mice, which were most pronounced at 6 months. Importantly, LRRK2 kinase inhibition reversed these effects in LRRK2G2019S mice, but had minimal effect in wild-type mice, suggesting that LRRK2 kinase inhibition is likely to reverse specific disease processes in G2019S mutation carriers. Additional work may be necessary to determine the potential effect in non-carriers.ConclusionsThis work supports a protective role of LRRK2 kinase inhibition in G2019S carriers and provides a rational workflow for systematic evaluation of brain-wide phenotypes in therapeutic development.

G2019S selective LRRK2 kinase inhibitor abrogates mitochondrial DNA damage

Pathogenic mutations in LRRK2 cause Parkinson’s disease (PD). The G2019S variant is the most common, which results in abnormally high kinase activity. Compounds that target LRRK2 kinase activity are currently being developed and tested in clinical trials. We recently found that G2019S LRRK2 causes mitochondrial DNA (mtDNA) damage and treatment with multiple classes of LRRK2 kinase inhibitors at concentrations associated with dephosphorylation of LRRK2 reversed mtDNA damage to healthy control levels. Because maintaining the normal function of LRRK2 in heterozygous G2019S LRRK2 carriers while specifically targeting the G2019S LRRK2 activity could have an advantageous safety profile, we explored the efficacy of a G2019S mutant selective LRRK2 inhibitor to reverse mtDNA damage in G2019S LRRK2 models and patient cells relative to non-selective LRRK2 inhibitors. Potency of LRRK2 kinase inhibition by EB-42168, a G2019S mutant LRRK2 kinase inhibitor, and MLi-2, a non-selective inhibitor, was determined by measuring phosphorylation of LRRK2 at Ser935 and/or Ser1292 using quantitative western immunoblot analysis. The Mito DNADX assay, which allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, was performed in parallel. We confirmed that EB-42168 selectively inhibits LRRK2 phosphorylation on G2019S LRRK2 relative to wild-type LRRK2. On the other hand, MLi-2 was equipotent for wild-type and G2019S LRRK2. Acute treatment with EB-42168 inhibited LRRK2 phosphorylation and also restored mtDNA damage to healthy control levels. We further investigated the relationship between LRRK2 kinase activity, mtDNA damage and mitophagy. Levels of mtDNA damage caused by G2019S LRRK2 were fully re-established within 2 h of a LRRK2 inhibitor wash out and recovery experiment, indicating the mtDNA damage phenotype is highly dynamic. G2019S LRRK2 mitophagy defects were not alleviated with LRRK2 kinase inhibition, suggesting that mitophagy is not mechanistically regulating LRRK2 kinase-mediated reversal of mtDNA damage in this acute timeframe. Abrogation of mtDNA damage with the mutant selective tool inhibitor EB-42168 demonstrates the potential of a precision medicine approach for LRRK2 G2019S PD. Levels of mtDNA damage may serve as a potential pharmacodynamic biomarker of altered kinase activity that could be useful for small molecule development and clinical trials.

Pharmacological Inhibition of LRRK2 Exhibits Neuroprotective Activity in Mouse Photothrombotic Stroke Model.

Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common cause of Parkinson's disease (PD). Interestingly, recent studies have reported an increased risk of stroke in patients with PD harboring LRRK2 mutations, but there is no evidence showing the functional involvement of LRRK2 in stroke. Here, we found that LRRK2 kinase activity was significantly induced in the Rose-Bengal (RB) photothrombosis-induced stroke mouse model. Interestingly, stroke infarct volumes were significantly reduced, and neurological deficits were diminished by pharmacological inhibition of LRRK2 kinase activity using MLi-2, a brain-penetrant LRRK2 kinase inhibitor. Immunohistochemical analysis showed p-LRRK2 level in stroke lesions, co-localizing with mitophagy-related proteins (PINK, Parkin, LC3B, cytochrome c), suggesting their involvement in stroke progression. Overlapping p-LRRK2 with cytochrome c/TUNEL/JC-1 (an indicator of mitochondrial membrane potential) puncta in RB photothrombosis indicated LRRK2-induced mitochondrial apoptosis, which was blocked by MLi-2. These results suggest that pharmacological inhibition of LRRK2 kinase activity could attenuate mitochondrial apoptosis, ultimately leading to neuroprotective potential in stroke progression. In conclusion, LRRK2 kinase activity might be neuro-pathogenic due to impaired mitophagy in stroke progression, and pharmacological inhibition of LRRK2 kinase activity could be beneficial in reducing the risk of stroke in patients with LRRK2 mutations.

A potential patient stratification biomarker for Parkinson´s disease based on LRRK2 kinase-mediated centrosomal alterations in peripheral blood-derived cells

Parkinson´s disease (PD) is a common neurodegenerative movement disorder and leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for disease intervention. However, the ability to stratify patients who will benefit from such treatment modalities based on shared etiology is critical for the success of disease-modifying therapies. Ciliary and centrosomal alterations are commonly associated with pathogenic LRRK2 kinase activity and can be detected in many cell types. We previously found centrosomal deficits in immortalized lymphocytes from G2019S-LRRK2 PD patients. Here, to investigate whether such deficits may serve as a potential blood biomarker for PD which is susceptible to LRKK2 inhibitor treatment, we characterized patient-derived cells from distinct PD cohorts. We report centrosomal alterations in peripheral cells from a subset of early-stage idiopathic PD patients which is mitigated by LRRK2 kinase inhibition, supporting a role for aberrant LRRK2 activity in idiopathic PD. Centrosomal defects are detected in R1441G-LRRK2 and G2019S-LRRK2 PD patients and in non-manifesting LRRK2 mutation carriers, indicating that they accumulate prior to a clinical PD diagnosis. They are present in immortalized cells as well as in primary lymphocytes from peripheral blood. These findings indicate that analysis of centrosomal defects as a blood-based patient stratification biomarker may help nominate idiopathic PD patients who will benefit from LRRK2-related therapeutics.

Identification of novel LRRK2 inhibitors by structure-based virtual screening and alchemical free energy calculation.

The Leucine-rich repeat kinase 2 (LRRK2) target has been identified as a promising drug target for Parkinson's disease (PD) treatment. This study focuses on optimizing the activity of LRRK2 inhibitors using alchemical relative binding free energy (RBFE) calculations. Initially, we assessed various free energy calculation methods across different LRRK2 kinase inhibitor scaffolds. The results indicate that alchemical free energy calculations are promising for prospective predictions on LRRK2 inhibitors, especially for the aminopyrimidine scaffold with an RMSE of 1.15 kcal mol-1 and Rp of 0.83. Following this, we optimized a potent LRRK2 kinase inhibitor identified from previous virtual screenings, featuring a novel scaffold. Guided by RBFE predictions using alchemical methods, this optimization led to the discovery of compound LY2023-001. This compound, with a [1,2,4]triazolo[5,6-b]indole scaffold, exhibited enhanced inhibitory activity against G2019S LRRK2 (IC50 = 12.9 nM). Molecular dynamics (MD) simulations revealed that LY2023-001 formed stable hydrogen bonds with Glu1948, and Ala1950 in the G2019S LRRK2 protein. Additionally, its phenyl substituents engage in strong electrostatic interactions with Lys1906 and van der Waals interactions with Leu1885, Phe1890, Val1893, Ile1933, Met1947, Leu1949, Leu2001, Ala2016, and Asp2017. Our findings underscore the potential of computational methods in the successful optimization of small molecules, offering important insights for the development of novel LRRK2 inhibitors.

Differential LRRK2 Signalling and Gene Expression in WT-LRRK2 and G2019S-LRRK2 Mouse Microglia Treated with Zymosan and MLi2.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause autosomal dominant Parkinson's disease (PD), with the most common causative mutation being the LRRK2 p.G2019S within the kinase domain. LRRK2 protein is highly expressed in the human brain and also in the periphery, and high expression of dominant PD genes in immune cells suggests involvement of microglia and macrophages in inflammation related to PD. LRRK2 is known to respond to extracellular signalling including TLR4, resulting in alterations in gene expression, with the response to TLR2 signalling through zymosan being less known. Here, we investigated the effects of zymosan, a TLR2 agonist and the potent and specific LRRK2 kinase inhibitor MLi-2 on gene expression in microglia from LRRK2-WT and LRRK2 p.G2019S knock-in mice by RNA-sequencing analysis. We observed both overlapping and distinct zymosan and MLi-2 mediated gene expression profiles in microglia. At least two candidate genome-wide association (GWAS) hits for PD, CathepsinB (Ctsb) and Glycoprotein-nmb (Gpnmb), were notably downregulated by zymosan treatment. Genes involved in inflammatory response and nervous system development were up and downregulated, respectively, with zymosan treatment, while MLi-2 treatment particularly exhibited upregulated genes for ion transmembrane transport regulation. Furthermore, we observed that the top twenty most significantly differentially expressed genes in LRRK2 p.G2019S microglia show enriched biological processes in iron transport and response to oxidative stress. Overall, these results suggest that microglial LRRK2 may contribute to PD pathogenesis through altered inflammatory pathways. Our findings should encourage future investigations of these putative avenues in the context of PD pathogenesis.

Inhibition of LRRK2 kinase activity rescues deficits in striatal dopamine physiology in VPS35 p.D620N knock-in mice

Dysregulation of dopamine neurotransmission profoundly affects motor, motivation and learning behaviors, and can be observed during the prodromal phase of Parkinson’s disease (PD). However, the mechanism underlying these pathophysiological changes remains to be elucidated. Mutations in vacuolar protein sorting 35 (VPS35) and leucine-rich repeat kinase 2 (LRRK2) both lead to autosomal dominant PD, and VPS35 and LRRK2 may physically interact to govern the trafficking of synaptic cargos within the endo-lysosomal network in a kinase-dependent manner. To better understand the functional role of VPS35 and LRRK2 on dopamine physiology, we examined Vps35 haploinsufficient (Haplo) and Vps35 p.D620N knock-in (VKI) mice and how their behavior, dopamine kinetics and biochemistry are influenced by LRRK2 kinase inhibitors. We found Vps35 p.D620N significantly elevates LRRK2-mediated phosphorylation of Rab10, Rab12 and Rab29. In contrast, Vps35 haploinsufficiency reduces phosphorylation of Rab12. While striatal dopamine transporter (DAT) expression and function is similarly impaired in both VKI and Haplo mice, that physiology is normalized in VKI by treatment with the LRRK2 kinase inhibitor, MLi-2. As a corollary, VKI animals show a significant increase in amphetamine induced hyperlocomotion, compared to Haplo mice, that is also abolished by MLi-2. Taken together, these data show Vps35 p.D620N confers a gain-of-function with respect to LRRK2 kinase activity, and that VPS35 and LRRK2 functionally interact to regulate DAT function and striatal dopamine transmission.

Inhibition of Parkinson's disease-related LRRK2 by type I and type II kinase inhibitors: Activity and structures.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial Parkinson's disease (PD) and a risk factor for the sporadic form. Increased kinase activity was shown in patients with both familial and sporadic PD, making LRRK2 kinase inhibitors a major focus of drug development efforts. Although much progress has been made in understanding the structural biology of LRRK2, there are no available structures of LRRK2 inhibitor complexes. To this end, we solved cryo-electron microscopy structures of LRRK2, wild-type and PD-linked mutants, bound to the LRRK2-specific type I inhibitor MLi-2 and the broad-spectrum type II inhibitor GZD-824. Our structures revealed an active-like LRRK2 kinase in the type I inhibitor complex, and an inactive DYG-out in the type II inhibitor complex. Our structural analysis also showed how inhibitor-induced conformational changes in LRRK2 are affected by its autoinhibitory N-terminal repeats. The structures provide a template for the rational development of LRRK2 kinase inhibitors covering both canonical inhibitor binding modes.

Leucine-rich repeat kinase 2 (LRRK2) inhibition upregulates microtubule-associated protein 1B to ameliorate lysosomal dysfunction and parkinsonism.

Mutations in LRRK2 (encoding leucine-rich repeat kinase 2 protein, LRRK2) are the most common genetic risk factors for Parkinson's disease (PD), and increased LRRK2 kinase activity was observed in sporadic PD. Therefore, inhibition of LRRK2 has been tested as a disease-modifying therapeutic strategy using the LRRK2 mutant mice and sporadic PD. Here, we report a newly designed molecule, FL090, as a LRRK2 kinase inhibitor, verified in cell culture and animal models of PD. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice and SNCA A53T transgenic mice, FL090 ameliorated motor dysfunctions, reduced LRRK2 kinase activity, and rescued loss in the dopaminergic neurons in the substantia nigra. Notably, by RNA-Seq analysis, we identified microtubule-associated protein 1 (MAP1B) as a crucial mediator of FL090's neuroprotective effects and found that MAP1B and LRRK2 co-localize. Overexpression of MAP1B rescued 1-methyl-4-phenylpyridinium induced cytotoxicity through rescuing the lysosomal function, and the protective effect of FL090 was lost in MAP1B knockout cells. Further studies may be focused on the in vivo mechanisms of MAP1B and microtubule function in PD. Collectively, these findings highlight the potential of FL090 as a therapeutic agent for sporadic PD and familial PD without LRRK2 mutations.

LRRK2 is involved in the chemotaxis of neutrophils and differentiated HL-60 cells, and the inhibition of LRRK2 kinase activity increases fMLP-induced chemotactic activity

BackgroundNeutrophils depend heavily on glycolysis for energy production under normal conditions. In contrast, neutrophils require energy supplied by mitochondrial oxidative phosphorylation (OXPHOS) during chemotaxis. However, the mechanism by which the energy supply changes from glycolysis to OXPHOS remains unknown. Leucine-rich repeat kinase 2 (LRRK2) is partially present in the outer mitochondrial membrane fraction. Lrrk2-deficient cells show mitochondrial fragmentation and reduced OXPHOS activity. We have previously reported that mitofusin (MFN) 2 is involved in chemotaxis and OXPHOS activation upon chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation in differentiated HL-60 (dHL-60) cells. It has been previously reported that LRRK2 binds to MFN2 and partially colocalizes with MFN2 at the mitochondrial membranes. This study investigated the involvement of LRRK2 in chemotaxis and MFN2 activation in neutrophils and dHL-60 cells.MethodsLrrk2 knockout neutrophils and Lrrk2 knockdown dHL-60 cells were used to examine the possible involvement of LRRK2 in chemotaxis. Lrrk2 knockdown dHL-60 cells were used a tetracycline-inducible small hairpin RNA (shRNA) system to minimize the effects of LRRK2 knockdown during cell culture. The relationship between LRRK2 and MFN2 was investigated by measuring the GTP-binding activity of MFN2 in Lrrk2 knockdown dHL-60 cells. The effects of LRRK2 kinase activity on chemotaxis were examined using the LRRK2 kinase inhibitor MLi-2.ResultsfMLP-induced chemotactic activity was reduced in Lrrk2 knockout neutrophils in vitro and in vivo. Lrrk2 knockdown in dHL-60 cells expressing Lrrk2 shRNA also reduced fMLP-induced chemotactic activity. Lrrk2 knockdown dHL-60 cells showed reduced OXPHOS activity and suppressed mitochondrial morphological change, similar to Mfn2 knockdown dHL-60 cells. The amount of LRRK2 in the mitochondrial fraction and the GTP-binding activity of MFN2 increased upon fMLP stimulation, and the MFN2 GTP-binding activity was suppressed in Lrrk2 knockdown dHL-60 cells. Furthermore, the kinase activity of LRRK2 and Ser935 phosphorylation of LRRK2 were reduced upon fMLP stimulation, and LRRK2 kinase inhibition by MLi-2 increased the migration to fMLP.ConclusionsLRRK2 is involved in neutrophil chemotaxis and the GTP-binding activity of MFN2 upon fMLP stimulation. On the other hand, the kinase activity of LRRK2 shows a negative regulatory effect on fMLP-induced chemotactic activity in dHL-60 cells.-J661noP-dBkFUp6aS7aWrVideo